The hibernation promoting factor of Betaproteobacteria Comamonas testosteroni cannot induce 100S ribosome formation but stabilizes 70S ribosomal particles.

Bacteria use several means to survive under stress conditions such as nutrient depletion. One such response is the formation of hibernating 100S ribosomes, which are translationally inactive 70S dimers. In Gammaproteobacteria (Enterobacterales), 100S ribosome formation requires ribosome modulation factor (RMF) and short hibernation promoting factor (HPF), whereas it is mediated by only long HPF in the majority of bacteria. Here, we investigated the role of HPFs of Comamonas testosteroni, which belongs to the Betaproteobacteria with common ancestor to the Gammaproteobacteria. C. testosteroni has two genes of HPF homologs of differing length (CtHPF-125 and CtHPF-119). CtHPF-125 was induced in the stationary phase, whereas CtHPF-119 conserved in many other Betaproteobacteria was not expressed in the culture conditions used here. Unlike short HPF and RMF, and long HPF, CtHPF-125 could not form 100S ribosome. We first constructed the deletion mutant of Cthpf-125 gene. When the deletion mutant grows in the stationary phase, 70S particles were degraded faster than in the wild strain. CtHPF-125 contributes to stabilizing the 70S ribosome. CtHPF-125 and CtHPF-119 both inhibited protein synthesis by transcription-translation in vitro. Our findings suggest that CtHPF-125 binds to ribosome, and stabilizes 70S ribosomes, inhibits translation without forming 100S ribosomes and supports prolonging life.

Variovorax durovernensis sp. nov., a novel species isolated from an infected prosthetic aortic graft in a human.

A novel bacterial strain, GSTT-20T was isolated from an infected, prosthetic endovascular graft explanted from a shepherd in London, United Kingdom. This strain was an aerobic, catalase-positive, oxidase-negative, Gram-stain-negative, motile, curved rod. It grew on blood agar, chocolate agar and MacConkey agar incubated at 37 °C in an aerobic environment after 48 h, appearing as yellow, mucoid colonies. Analysis of the complete 16S rRNA gene sequence showed closest similarity to Variovorax paradoxus with 99.6 % identity and Variovorax boronicumulans with 99.5 % identity. Phylogenetic analysis of the 16S rRNA gene sequence and phylogenomic analysis of single nucleotide polymorphisms within 1530 core genes showed GSTT-20T forms a distinct lineage in the genus Variovorax of the family Comamonadaceae. In silico DNA-DNA hybridization assays against GSTT-20T were estimated at 32.1 % for V. boronicumulans and 31.9 % for V. paradoxus. Genome similarity based on average nucleotide identity was 87.50 % when comparing GSTT-20T to V. paradoxus. Based on these results, the strain represented a novel species for which the name Variovorax durovernensis sp. nov. was proposed. The type strain is GSTT-20T (NCTC 14621T=CECT 30390T).

Scleromatobacter humisilvae gen. nov., sp. nov., a novel bacterium isolated from oak forest soil.

A novel bacterial strain, designated BS-T2-15T, isolated from forest soil in close proximity to decaying oak wood, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences as well as phylogenomic analyses based on coding sequences of 340 concatenated core proteins indicated that strain BS-T2-15T forms a distinct and robust lineage in the Rubrivivax-Roseateles -Leptothrix-Azohydromonas -Aquincola-Ideonella branch of the order Burkholderiales. The amino acid identity and the percentage of conserved proteins between the genome of strain BS-T2-15T and genomes of closely related type strains ranged from 64.27 to 66.57% and from 40.89 to 49.27 %, respectively, providing genomic evidence that strain BS-T2-15T represents a new genus. Its cells are Gram-stain-negative, aerobic, motile by a polar flagellum, rod-shaped and form incrusted white to ivory colonies. Optimal growth is observed at 20-22 °C, pH 6 and 0% NaCl. The predominant fatty acids of strain BS-T2-15T are C16 : 1 ω7c, C16 : 0 and C14 : 0 2-OH. Its polar lipid profile consists of a mixture of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol and its main respiratory quinone is ubiquinone 8. The estimated size of its genome is 6.28 Mb with a DNA G+C content of 69.56 mol%. Therefore, on the basis of phenotypic and genotypic properties, the new strain BS-T2-15T represents a novel genus and species for which the name Scleromatobacter humisilvae gen. nov., sp. nov., is proposed. The type strain is BS-T2-15T (DSM 113115T=UBOCC-M-3373T).

Chromobacterium alticapitis sp. nov. and Chromobacterium sinusclupearum sp. nov. isolated from wild cranberry bogs in the Cape Cod National Seashore, USA.

Two non-pigmented strains in the genus Chromobacterium, MWU14-2602T and MWU13-2610T, were isolated from wild cranberry bogs in the Cape Cod National Seashore, USA. The isolates were characterized by genomic and phenotypic analyses, the results of which indicated that they represent two novel species. Based on total genome sequences, the closest relatives were in the Chromobacterium amazonense group, which includes the recently described Chromobacterium paludis. Whole genome sequences were compared by genome blast distance phylogeny, digital DNA-DNA hybridization and average nucleotide identity analyses with each other and with the type strains of their nearest species. MWU14-2602T and MWU13-2610T fell well below the accepted cutoff values for species relatedness, clearly indicating that they represent novel species. Although little is known about these organisms in situ, under laboratory conditions, MWU13-2610T produced a modest amount of HCN and was strongly positive for exoprotease activity, whereas MWU14-2602T did not produce HCN or exoproteases. The predominant fatty acids for both isolates were summed C16 : 1ω7cis/C16 : 1ω6cis. Both isolates produced siderophores and pyomelanin pigment on rich media, and neither was haemolytic on sheep blood agar. We propose the names Chromobacterium alticapitis sp. nov. (type strain MWU14-2602T=ATCC TSD 260T=CCOS 1979T) and Chromobacterium sinusclupearum sp. nov. (type strain MWU13-2610T=ATCC TSD-259T=CCOS 1981T) for these taxa.

Lifestyles Shape the Cytochrome P450 Repertoire of the Bacterial Phylum Proteobacteria.

For the last six decades, cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, have been under the spotlight due to their regio- and stereo-selective oxidation activities, which has led to the exploration of their applications in almost all known areas of biology. The availability of many genome sequences allows us to understand the evolution of P450s in different organisms, especially in the Bacteria domain. The phenomenon that "P450s play a key role in organisms' adaptation vis a vis lifestyle of organisms impacts P450 content in their genome" was proposed based on studies on a handful of individual bacterial groups. To have conclusive evidence, one must analyze P450s and their role in secondary metabolism in species with diverse lifestyles but that belong to the same category. We selected species of the phylum Proteobacteria classes, Alpha, Beta, Gamma, Delta, and Epsilon, to address this research gap due to their diverse lifestyle and ancient nature. The study identified that the lifestyle of alpha-, beta-, gamma-, delta-, and epsilon-proteobacterial species profoundly affected P450 profiles in their genomes. The study determined that irrespective of the species associated with different proteobacterial classes, pathogenic species or species adapted to a simple lifestyle lost or had few P450s in their genomes. On the contrary, species with saprophytic or complex lifestyles had many P450s and secondary metabolite biosynthetic gene clusters. The study findings prove that the phenomenon mentioned above is factual, and there is no link between the number and diversity of P450s and the age of the bacteria.

Molecular diversity of rhizobia-nodulating native Mimosa of Brazilian protected areas.

Symbiotic Paraburkholderia have been increasingly studied in the past 20 years, especially when associated with Mimosa; however, studies with native/endemic species are still scarce. In this study, thirty strains were isolated from root nodules of native Mimosa paranapiacabae and M. micropteris in two locations of the Campos Gerais. The BOX-PCR fingerprinting revealed high genomic diversity, and the 16S rRNA phylogeny clustered the strains in three distinct groups (GI, GII, GIII), with one strain occupying an isolated position. Phylogenetic analysis with four concatenated housekeeping genes (atpD + gltB + gyrB + recA) confirmed the same clusters of 16S rRNA, and the closest species were P. nodosa BR 3437T and P. guartelaensis CNPSo 3008T; this last one isolated from another Mimosa species of the Campos Gerais. The phylogenies of the symbiotic genes nodAC and nifH placed all strains in a well-supported branch with the other species of the symbiovar mimosae. The phylogenetic analyses indicated that the strains represent novel lineages of sv. mimosae and that endemic Mimosa coevolved with indigenous Paraburkholderia in their natural environments.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Aquabacterium soli sp. nov., a novel bacterium isolated from soil under the long-term application of bifenthrin.

Strain SJQ9T, an aerobic bacterium isolated from a soil sample collected in Shanghai, PR China, was characterized using a polyphasic approach. It grew optimally at pH 7.0, 30-35 °C and in the presence of 1 % (w/v) NaCl. A comparative analysis of 16S rRNA gene sequences showed that strain SJQ9T fell within the genus Aquabacterium. The closest phylogenetic relatives of strain SJQ9T were Aquabacterium citratiphilum DSM 11900T (98.6 % sequence similarity) and Aquabacterium commune DSM 11901T (96.4 %). Cells of the strain were Gram-stain-negative, motile, non-spore-forming, rod-shaped and positive for oxidase activity and negative for catalase. The chemotaxonomic properties of strain SJQ9T were consistent with those of the genus Aquabacterium: the major fatty acid was summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). The isoprenoid quinone was Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 65.7 mol%. Strain SH9T exhibited a DNA-DNA relatedness level of 34±2 % with A. citratiphilum DSM 11900T and 28±3 % with A. commune DSM 11901T. Based on the obtained data, strain SJQ9T represents a novel species of the genus Aquabacterium, for which the name Aquabacterium soli sp. nov. is proposed. The type strain is SJQ9T (=JCM 33106T=CCTCC AB 2018284T).

Ramlibacter algicola sp. nov., isolated from a freshwater alga Cryptomonas obovoidea.

A Gram-stain-negative, strictly aerobic, catalase-negative, oxidase-positive and non-motile rod-shaped bacterium, designated strain CrO1T, was isolated from a freshwater alga Cryptomonas obovoidea in the Nakdong river of South Korea. Colonies of CrO1T were white, convex and circular and growth was observed at 25-40 °C (optimum, 37 °C) and pH 6.0-9.0 (optimum, pH 7) and in the presence of 0-0.5 % (w/v) NaCl (optimum, 0 %). CrO1T contained C16 : 0, summed feature 5 (comprising C18 : 0ante and/or C18 : 2ω6,9c), C18 : 0, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) as the major cellular fatty acids (>5 %) and ubiquinone-8 as the sole respiratory quinone. Phosphatidylethanolamine was detected as the major polar lipid. The DNA G+C content of CrO1T, calculated from the whole genome sequence was 69.6 mol%. CrO1T was most closely related to Ramlibacter humi 18x22-1T with a 97.6 % 16S rRNA sequence similarity and shared less than 97.4 % 16S rRNA sequence similarities with other type strains. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences revealed that CrO1T formed a distinct phyletic lineage within the genus Ramlibacter. On the basis of the results of phenotypic, chemotaxonomic and molecular analysis, CrO1T clearly represents a novel species of the genus Ramlibacter, for which the name Ramlibacter algicola sp. nov. is proposed. The type strain is CrO1T (=KACC 19926T=JCM 33302T).

Molecular mechanism of sulfur chemolithotrophy in the betaproteobacterium Pusillimonas ginsengisoli SBSA.

Chemolithotrophic sulfur oxidation represents a significant part of the biogeochemical cycling of this element. Due to its long evolutionary history, this ancient metabolism is well known for its extensive mechanistic and phylogenetic diversification across a diverse taxonomic spectrum. Here we carried out whole-genome sequencing and analysis of a new betaproteobacterial isolate, Pusillimonas ginsengisoli SBSA, which is found to oxidize thiosulfate via the formation of tetrathionate as an intermediate. The 4.7 Mb SBSA genome was found to encompass a soxCDYZAXOB operon, plus single thiosulfate dehydrogenase (tsdA) and sulfite : acceptor oxidoreductase (sorAB) genes. Recombination-based knockout of tsdA revealed that the entire thiosulfate is first converted to tetrathionate by the activity of thiosulfate dehydrogenase (TsdA) and the Sox pathway is not functional in this bacterium despite the presence of all necessary sox genes. The ∆soxYZ and ∆soxXA knockout mutants exhibited a wild-type-like phenotype for thiosulfate/tetrathionate oxidation, whereas ∆soxB, ∆soxCD and soxO::KanR mutants only oxidized thiosulfate up to tetrathionate intermediate and had complete impairment in tetrathionate oxidation. The substrate-dependent O2 consumption rate of whole cells and the sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, indicated that glutathione plays a key role in SBSA tetrathionate oxidation. The present findings collectively indicate that the potential glutathione : tetrathionate coupling in P. ginsengisoli involves a novel enzymatic component, which is different from the dual-functional thiol dehydrotransferase (ThdT), while subsequent oxidation of the sulfur intermediates produced (e.g. glutathione : sulfodisulfane molecules) may proceed via the iterative action of soxBCD .

Chromobacterium paludis sp. nov., a novel bacterium isolated from a Chesapeake Bay marsh.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1T and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium. Genomic sequencing of IIBBL 257-1T revealed a genome of 4.27 Mb, with a G+C content of 63.6 %. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1T was most closely related to C. amazonense and C. phragmitis. Comparison of the IIBBL 257-1T genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30 %, respectively, for both. These results demonstrate that IIBBL 257-1T and IIBBL 257-2 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1T (=NRRL B-65555T=JCM 33770T).

Saezia sanguinis gen. nov., sp. nov., a Betaproteobacteria member of order Burkholderiales, isolated from human blood.

The taxonomic position of an unknown bacterial strain designated CNM695-12, isolated from the blood of an immunocompromised subject, was investigated via phenotypic, chemotaxonomic, genotypic and genomic analyses. Bacterial cells were determined to be Gram-stain-negative bacilli, aerobic, non-motile and non-spore-forming. The strain showed catalase activity but no oxidase activity. Optimal growth occurred at 37 °C, pH 7 and with 0-1 % NaCl. C16 : 0, summed feature 8 (comprising C18 : 1ω7c /C18:1 ω6c), and C18 : 1ω9c were the most abundant fatty acids, and ubiquinone 8 was the major respiratory quinone. The polar lipids present included phosphatidylglycerol, phosphatidylethanolamine and other aminophospholipids. The 16S rRNA gene sequence showed approximately 93.5 % similarity to those of different species with validly published names within the order Burkholderiales (e.g. Leptothrix mobilis Feox-1T, Aquabacterium commune B8T , Aquabacterium citratiphilum B4T and Schlegelella thermodepolymerans K14T). Phylogenetic analyses based on 16S rRNA gene sequences and concatenated alignments including the sequences for 107 essential proteins, revealed the strain to form a novel lineage close to members of the family Comamonadaceae. The highest average nucleotide identity and average amino acid identity values were obtained with Schlegelella thermodepolymerans K14T (69.6 and 55.7 % respectively). The genome, with a size of 3.35 Mb, had a DNA G+C content of 52.4 mol% and encoded 3056 predicted genes, 3 rRNA, 1 transfer-messengerRNA and 51 tRNA. Strain CNM695-12 thus represents a novel species belonging to a novel genus within the order Burkholderiales, for which the name Saezia sanguinis gen. nov., sp. nov. is proposed. The type strain is CNM695-12T (=DSM 104959T=CECT 9208T).

Orrella amnicola sp. nov., isolated from a freshwater river, reclassification of Algicoccus marinus as Orrella marina comb. nov., and emended description of the genus Orrella.

A novel Gram-negative, aerobic, non-motile, ovoid to rod-shaped bacterium, designated NBD-18T, was isolated from a freshwater river in Taiwan. Optimal growth occurred at 30 °C, at pH 6 and in the absence of NaCl. The predominant fatty acids of strain NBD-18T were C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 0 cyclo and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidyldimethylethanolamine. The major polyamine was putrescine. The major isoprenoid quinone was Q-8. The genomic DNA G+C content of strain NBD-18T was 50.9 %. Strain NBD-18T was most closely related to Orrella dioscoreae LMG 29303T and Algicoccus marinus HZ20T at a 16S rRNA gene sequence similarity of 97.7 %. 16S rRNA gene sequence similarity between O. dioscoreae LMG 29303T and A. marinus HZ20T was 97.7 %. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set indicated that strain NBD-18T, O. dioscoreae LMG 29303T and A. marinus HZ20T are affiliated with the same genus. Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values among these three strains supported that they belong to the same genus and that strain NBD-18T represents a novel species. Thus, A. marinus HZ20T should be reclassified as Orrella marina comb. nov. based on the rules for priority of publication and validation. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain NBD-18T represents a novel species in the genus Orrella, for which the name Orrella amnicola sp. nov. is proposed. The type strain is NBD-18T (=BCRC 81197T=LMG 31338T).

Ideonella livida sp. nov., isolated from a freshwater lake.

A novel bacterial strain, designated TBM-1T, isolated from a freshwater lake in Taiwan, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain TBM-1T formed a phylogenetic lineage in the genus Ideonella. Analysis of 16S rRNA gene sequences showed that strain TBM-1T was most closely related to Ideonella dechloratans CCUG 30898T with 98.4 % sequence similarity. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain TBM-1T and closely related strains of the genus Ideonella were 74.4-77.5 %, 69.7-75.4 % and 19.8-21.8 %, respectively, supporting that strain TBM-1T represents a novel species of the genus Ideonella. Cells were Gram-stain-negative, motile by means of a single polar flagellum, rod-shaped and formed blue colonies. Optimal growth occurred at 30 °C, pH 6 and 0 % NaCl. The predominant fatty acids of strain TBM-1T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two uncharacterized aminophospholipids and two uncharacterized phospholipids. The main polyamine was putrescine. The major isoprenoid quinone was Q-8. The estimated genome size was 5.26 Mb, with an average G+C content of 70.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain TBM-1T should be classified in a novel species of the genus Ideonella, for which the name Ideonella livida sp. nov. is proposed. The type strain is TBM-1T (=BCRC 81199T =LMG 31339T).

Massilia arenosa sp. nov., isolated from the soil of a cultivated maize field.

Strain MC02T, a Gram-stain-negative, rod-shaped bacterium, was isolated from field soil collected from California, USA. To examine if MC02T represents a novel species, we compared its colony morphology, 16S rRNA gene and whole genome sequence, and its metabolic phenotype using Biolog GenIII and MALDI-TOF analyses compared to reference strains. Based on 16S rRNA gene and whole genome sequencing, MC02T belongs to the genus Massilia and Massilia agri K-3-1T is the most similar strain with 96.97 % 16S rRNA gene sequence identity. MALDI-TOF analysis revealed that Massilia aerilata DSM19289T is the closest match, but the similarity score was much lower than the ≥1.7 threshold for a reliable identification at the genus level. The predominant fatty acids were summed feature 3 (C16 : 1⍵7c and/or C16 : 1⍵6c; 49.07 %) and C16 : 0 (30.01 %). The genome is 5.02 Mbp and the G+C content is 66.2 mol%. Whole genome comparisons to the closest related strains revealed an average amino acid identity value of 67.4 %, an OrthoANI similarity of 77.1 %, and a DNA-DNA-hybridization probability ≥70 %, confirming that MC02T represents a novel species. Strain MC02T can grow at pH 6 but not at pH 5, and a salt concentration of ≥1 % inhibits its growth. In contrast to other Massilia strains, MC02T can utilize turanose, inosine and l-serine. The genome of MC02T shows putative endophyte genes such as a nitrate reductase, several phosphatases, and biotin biosynthesis genes, 26 flagellar motility genes and 14 invasion and intracellular resistance genes. Based on its metabolic, physiological and genomic characteristics, we propose that strain MC02T (NRRL B-65554T=ATCC TSD-200T=LMG 31737T) represents a novel species of the genus Massilia with the name Massilia arenosa sp. nov.

Aquabacterium pictum sp. nov., the first aerobic bacteriochlorophyll a-containing fresh water bacterium in the genus Aquabacterium of the class Betaproteobacteria.

A strictly aerobic, bacteriochlorophyll a-containing betaproteobacterium, designated strain W35T, was isolated from a biofilm sampled at Tama River in Japan. The non-motile and rod-shaped cells formed pink-beige pigmented colonies on agar plates containing organic compounds, and showed an in vivo absorption maximum at 871 nm in the near-infrared region, typical for the presence of bacteriochlorophyll a. The new bacterial strain is Gram-negative, and oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain W35T was closely related to species in the genus Aquabacterium. The closest phylogenetic relatives of strain W35T were Aquabacterium commune B8T (97.9 % sequence similarity), Aquabacterium citratiphilum B4T (97.2 %) and Aquabacterium limnoticum ABP-4T (97.0 %). The major cellular fatty acids were C16  :  1ω7c (50.4 %), C16  :  0 (22.7 %), summed feature 8 (C18  :  1ω7c/C18  :  1ω6c; 9.7 %), C18  :  3ω6c (5.5 %), C12  :  0 (5.3 %) and C10  :  0 3OH (2.7 %). The respiratory quinone was ubiquinone-8. Predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the genomic DNA was 70.4 mol% (genome data) and 71.4 mol% (HPLC). The genome size of strain W35T is 6.1 Mbp and average nucleotide identity analysis indicated genome similarities of strain W35T and related Aquabacterium type strains to be 78-79 %. The results of polyphasic comparisons showed that strain W35T was clearly distinguishable from other members of the genus Aquabacterium. Therefore, we propose a new species in the genus Aquabacterium, namely, Aquabacterium pictum sp. nov. The type strain is W35T (=DSM 106757T=NBRC 111963T). The description of the genus Aquabacterium is also emended.

Rubrivivax albus sp. nov., isolated from a freshwater pond.

Strain ICH-3T, isolated from a freshwater pond in Taiwan 9ROC), was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that ICH-3T is affiliated with the species in the genus Rubrivivax. ICH-3T was most closely related to Rubrivivax benzoatilyticus JA2T and Rubrivivax gelatinosus NCIB 8290T with 97.5 and 97.4 % 16S rRNA gene sequence similarity. The average nucleotide identity and digital DNA-DNA hybridization identity between ICH-3T and the two closely related strains were 77.3 and 20.9-21.0 %, respectively, indicating that ICH-3T represents a novel species of the genus Rubrivivax. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed white colonies. Optimal growth occurred at 30 °C, pH 7.5-8.0 and with 0.5 % NaCl. The major fatty acids (>20 %) of ICH-3T were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an uncharacterized aminophospholipid and two uncharacterized phospholipids. The major isoprenoid quinone was Q-8. The genomic DNA G+C content of ICH-3T was 70.3 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain ICH-3T should be classified as representing a novel species of the genus Rubrivivax, for which the name Rubrivivax albus sp. nov. is proposed. The type strain is ICH-3T (=BCRC 81155T=LMG 30930T=KCTC 62866T).

Aquabacterium lacunae sp. nov., isolated from a freshwater pond.

A novel bacterial strain, designated KMB7T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, motile by means of a single polar flagellum, rod-shaped and formed cream colonies. Optimal growth occurred at 25 °C, pH 7, and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain KMB7T is affiliated with species in the genus Aquabacterium. The 16S rRNA gene sequence similarity indicated that strain KMB7T is closely related to species within the genus Aquabacterium (95.2-97.6 % sequence similarity) and is most similar to A. fontiphilum CS-6T (97.6 %), followed by A. parvum B6T (97.5 %). The average nucleotide identity and digital DNA-DNA hybridization identity between strain KMB7T and the closely related strains were 74.6-78.0 % and 19.0-21.2 %, respectively. The major fatty acids of strain KMB7T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol and four unidentified phospholipids. The sole isoprenoid quinone was ubiquinone-8 (Q-8). Genomic DNA G+C content of strain KMB7T was 65.4 %. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain KMB7T should be classified in a novel species of the genus Aquabacterium, for which the name Aquabacterium lacunae sp. nov. is proposed. The type strain is KMB7T (=BCRC 81156T=LMG 30924T=KCTC 62867T).

Comparative genome sequence analysis of several species in the genus Tepidimonas and the description of a novel species Tepidimonas charontis sp. nov.

We performed high-quality genome sequencing of eight strains of the species of the genus Tepidimonas and examined the genomes of closely related strains from the databases to understand why Tepidimonas taiwanensis is the only strain of this genus that utilizes glucose and fructose for growth. We found that the assimilation of these hexoses by T. taiwanensis was due to the presence of two transporters that are absent in all other genomes of strains of members of the genus Tepidimonas examined. Some strains lack genes coding for glucokinase, but the Embden-Meyerhof-Parnas pathway appears to be otherwise complete. The pentose phosphate pathway has a complete set of genes, but genes of the Entner-Doudoroff pathway were not identified in the genomes of any of the strains examined. Genome analysis using average nucleotide identity (ANIb), digital DNA-DNA hybridization (dDDH), average amino acid identity (AAI) and phylogenetic analysis of 400 conserved genes was performed to assess the taxonomic classification of the organisms. Two isolates of the genus Tepidimonas from the hot spring at São Pedro do Sul, Portugal, designated SPSP-6T and SPSPC-18 were also examined in this study. These organisms are mixotrophic, have an optimum growth temperature of about 50 ºC, utilize several organic acids and amino acids for growth but do not grow on sugars. Distinctive phenotypic, 16S rRNA gene sequence and genomic characteristics of strains SPSP-6T and SPSPC-18 lead us to propose a novel species based on strain SPSP-6T for which we recommend the name Tepidimonas charontis sp. nov. (=CECT 9683T=LMG 30884T).

Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing.

Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with 13C-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of 13C was detected in RNA isolated from BACTRAPs loaded with 13C-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable 13C incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with 13C and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no 13C label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.